Proteomics & Genomics
Dr. Vikash Kumar Dubey
Lecture 21 : Midterm examination
Total marks 50
1
What may be possible causes of wrong identification of protein using peptide
mass finger printing?
3
2
A solution containing aspartic acid (PI=2.98), glycine (PI=5.97), leucine
(PI=5.98), and lysine (PI=9.74) in a pH 3.0 citrate buffer was applied to a
Dowex-50 column equilibrated with the same buffer. The column was then
eluted with increasing NaCl concentration. In what order will the amino acids
elute from the column?
Write short notes on
a) Magnetic sector analyzer of Mass spectrometer
b) TOF analyzer of Mass spectrometer explain reflectron mode of TOF
3
4
What is immobilized metal affinity chromatography (IMAC)? Explain why
Ni-NTA (NTA: -Nitrilotriacetic acid) is preferred over Ni-IDA (IDA:
iminodiacetic acid). Explain why Ni-NTA chromatography is not done in
acidic pH range. What are different methods of elution of protein from NiNTA?
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5
What is activity staining of enzyme? Explain why activity staining is not a
preferred method when you use isoelectric focusing.
5
6
Size exclusion chromatography: Native-galactosidase elutes at 20ml. what
elution volume do you expect for denatured -galactosidase under identical
experimental conditions.
5
7.
Write short note on following affinity purification methods.
1. His tag
2. GST-tag
How affinity tag is removed from the protein after purification?
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8
Reply following multiple choice questions
3
3
1. PAGE is a technique of separation of ______ based on their molecular
weight and charge density.
A.
B.
C.
D.
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Proteins
DNA
RNA
Amino acids
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Proteomics & Genomics
Dr. Vikash Kumar Dubey
2. In SDS PAGE proteins are separated only based on their –
A. Charge
B. Molecular weight
C. pI
D. Hydrophobicity
3. The crosslinking agent used for PAGE gel isA. Acrylamide
B. TEMED
C. APS
D. N,N’ methylene bisacrylamide
4. The resolving gel has a pH of A. 8.8
B. 6.8
C. 7.8
D. 9.8
5. Acrylamide concentration of the stacking gel is always _______ than
that of the resolving gel.
A. Greater
B. Lesser
C. Same
D. May be greater or lesser
6. The dye used to track the migration of proteins in the gel isA. Coomassie Brilliant Blue
B. Silver Stain
C. Beta mercaptoethanol
D. Bromo phenol blue
7. SDS imparts _______ charge to the proteins.
A. Negative
B. Positive
C. Neutral
D. Does not effect the charge of the proteins
8. The compound added to disrupt the disulfide bonds present in the
proteins isA. TEMED
B. APS
C. Beta mercaptoethanol
D. SDS
9. The pH of the running buffer is
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Proteomics & Genomics
A.
B.
C.
D.
Dr. Vikash Kumar Dubey
6.3
7.3
8.3
9.3
10. Acrylamide percentage present in stacking gel is –
A. 4-5%
B. 6-7%
C. 8-9%
D. 2-3%
11. Based on what property proteins are separated in 2D PAGE?
A. Molecular Weight.
B. Isoelectric point
C. Both.
D. None.
12. For creating pH gradient in IEF ________ are used.
A. Ampholytes.
B. SDS.
C. Polyacrylamides.
D. Detergents.
13. pI is the pH at which proteins have ________ charge.
A. Positive.
B. Negative.
C. Zero.
D. Minimum.
14. After IEF ______ is added to the proteins to denature them and impart
equal negative charges to the proteins.
A. Polyacrylamide.
B. SDS.
C. Ampholyte.
D. Coomassie Blue.
15. In 2D PAGE second electrophoresis is done at ______ degree angle
relative to the first one.
A. 60
B. 180
C. 90
D. 30
16 Dimensinal gel electrophoresis involves the following two techniquesA. Flourescence spectroscopy and HPLC
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Proteomics & Genomics
Dr. Vikash Kumar Dubey
B. SDS-PAGE and Ion exchange chromatography
C. SDS-PAGE and IEF
D. Affinity chromatography and size exclusion chromatography
17. The component of the gel which maintains the pH gradient is calledA. Ampholytes
B. Aminoacids
C. Zwitter ions
D. Poly acrylamide
18. In pH gradient of the gel, low pH belongs toA. More negative charge
B. More positive charge
C. Neutral
D. None of the above
19. In 2DE the final gel separates the proteins on the basis ofA. pI and MW
B. pH and Molarity
C. Charge and MW
D. Charge and pI
20.. IEF is widely applied forA. drug testing
B. protein purification
C. protein identification
D. protein digestion
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Proteomics & Genomics
Dr. Vikash Kumar Dubey
Submit your assignment to course developer by e-mail;
you will get an e-mail reply with grading and feedback in
a week time.
Answers to multiple choice questions
1. A
2. B
3. D
4. A
5. B
6. D
7. A
8. C
9. C
10. A
14. C
15. A
16. C
17. B
18. C
19. C
20. A
21. B
22. A
23. C
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